Population Of Stem Cell-like Cells Research Articles

Regulation of epidermal proliferation and hair follicle cycling by synthetic photostable retinoid EC23.

Retinoid signaling is an important regulator of the epidermis and skin appendages. Therefore, synthetic retinoids have been developed for therapeutic use for skin disorders such as psoriasis and acne. In previous studies, we showed how the photostable retinoid EC23 induces neuronal differentiation in stem cell-like cell populations, and here, we aim to investigate its ability to influence epidermal and hair follicle growth. EC23 influence on skin biology was investigated initially in cultures of monolayer keratinocytes and three-dimentional in vitro models of skin, and finally in in vivo studies of mice back skin. EC23 induces keratinocyte hyperproliferation in vitro and in vivo, and when applied to mouse skin increases the number of involucrin-positive suprabasal cell layers. These phenotypic changes are similar in skin treated with the natural retinoid all-trans retinoic acid (ATRA); however, EC23 is more potent; a tenfold lower dose of EC23 is sufficient to induce epidermal thickening, and resulting hyperproliferation is sustained for a longer time period after first dose. EC23 treatment resulted in a disorganized stratum corneum, reduced cell surface lipids and compromised barrier, similar to ATRA treatment. However, EC23 induces a rapid telogen to anagen transition and hair re-growth in 6-week-old mice with synchronously resting back skin follicles. The impact of EC23 on the hair cycle was surprising as similar results have not been seen with ATRA. These data suggest that synthetic retinoid EC23 is a useful tool in exploring the turnover and differentiation of cells and has a potent effect on skin physiology.

Sci-Seq of Human Fetal Salivary Tissue Introduces Human Transcriptional Paradigms and a Novel Cell Population.

Multiple pathologies and non-pathological factors can disrupt the function of the non-regenerative human salivary gland including cancer and cancer therapeutics, autoimmune diseases, infections, pharmaceutical side effects, and traumatic injury. Despite the wide range of pathologies, no therapeutic or regenerative approaches exist to address salivary gland loss, likely due to significant gaps in our understanding of salivary gland development. Moreover, identifying the tissue of origin when diagnosing salivary carcinomas requires an understanding of human fetal development. Using computational tools, we identify developmental branchpoints, a novel stem cell-like population, and key signaling pathways in the human developing salivary glands by analyzing our human fetal single-cell sequencing data. Trajectory and transcriptional analysis suggest that the earliest progenitors yield excretory duct and myoepithelial cells and a transitional population that will yield later ductal cell types. Importantly, this single-cell analysis revealed a previously undescribed population of stem cell-like cells that are derived from SD and expresses high levels of genes associated with stem cell-like function. We have observed these rare cells, not in a single niche location but dispersed within the developing duct at later developmental stages. Our studies introduce new human-specific developmental paradigms for the salivary gland and lay the groundwork for the development of translational human therapeutics.

Therapeutic and clinical propositions of Mesenchymal Stem Cells(MSCs) focusing on recent trial updates

The mesenchymal stroma harbours considerable population of stem cell-like cells with differentiation and self-renewal abilities that originate from several sources like amniotic fluid, Wharton’s jelly, umbilical cord etc. Mesenchymal Stem Cells (MSCs) are most commonly found in the perivascular niche. This multipotent progenitor cells have the ability to differentiate into mesodermal cell types such as adipocytes, chondrocytes and osteocytes. MSCs can also exert significant immunosuppressive and anti-inflammatory effects by interacting with lymphocytes from both innate and adaptive immune system. MSCs of fetal origins can go through same processes as MSCs derived from elsewhere. Before senescence, they make more cell divisions than adult MSCs of bone marrow or adipose tissue. The propitious properties of mesenchymal stem cells (MSCs), such as their ability to differentiate into diverse cell lineages and their regenerative properties, have intrigued researchers, whose work has provided fascinating perspectives on cell-based therapies for wide range of diseases. MSCs have a high potential for replication in vitro. International Society for Cellular Therapy (ISCT)-based MSC isolation has resulted in heterogeneous, nonclonal stromal cell cultures containing stem cells with various multipotent characteristics, committed progenitors, and differentiated cells. Nonclonal stromal cultures which are derived from bone marrow and other tissues are presently used as sources of putative MSCs for therapeutic aims. Also, MSCs have potential to endogenously repair as well as reduce the inflammatory responses that may lead to decreased morbidity and mortality rate of COVID-19. Herein, the MSC-based clinical prospects, clarifies, the recent clinical findings, therapeutic effects of MSC and clinical trials.

How do signaling and transcription factors regulate both axis elongation and Hox gene expression along the anteroposterior axis?

In vertebrates, vertebral primordia, called somites, are formed from the head to the tail along the anteroposterior axis of the body during development. The presomitic mesoderm (PSM), which differentiates into somites, is formed by continuous supply of new cells derived from the caudal lateral epiblast (CLE), resulting in body axis posterior elongation. Previous studies of mutants identified genes for posterior extension and vertebral patterning along the anteroposterior axis. Hox gene has been extensively investigated for its expression pattern and transcriptional regulation. In recent years, to elucidate the mechanism that controls the expression patterns of Hox genes, researchers have not only searched for enhancer regions and the transcription factors that bind to them but have also investigated chromatin structure, epigenetics and non-coding RNA associated with Hox gene expression. These new findings reveal that the previously identified genes essential for posterior body axis elongation of the embryo determine positional information along the anteroposterior axis by induction of Hox genes via enhancer regions. In this review, we focus on genes that control posterior elongation and vertebral patterning along the anteroposterior axis in the PSM and CLE. We first describe the mechanism of maintenance of the stem cell-like cell populations at the CLE, which is essential for the posterior elongation of the embryo. Next, the factors involved in posterior region formation and patterning of the vertebra are described. Finally, we discuss the regulatory mechanism of Hox gene expression and the mechanism that is responsible for the differences in skeletal pattern between species.

LIVER CANCER STEM CELLS

Recent studies have shown that many tumours are containing small population of stem cell-like cells, in other words, cancer stem cells. The rapid development of the cancer stem cell field has allowed the scientists and the clinicians to focus on another challenge, which targets cancer stem cells for new therapeutic strategies. Studies showed that novel therapeutic approaches on the selective targeting of cancer stem cells might be extremely successful for prevention of invasion, metastasis, and the relapses of tumors. Liver cancer is the 5th most common cancer type. It has also been reported as the third most common cause of cancer related death. Recently, progression of hepatocellular carcinoma is thought to be driven by cancer stem cells. There are tremendous efforts to clarify the features of liver cancer stem cells, to identify them within other tumor cells, and finally to develop the new therapeutic models that target those cells. This review summarizes the features of liver cancer stem cells, and its importance for therapeutic approaches.

PO-482 Efficacy of inhibition of BAD Ser99 phosphorylation by a novel small molecule in cisplatin resistant ovarian cancer

IntroductionHuman BAD (hBAD) is a pro-apoptotic Bcl-2 family member, whose apoptotic functions can be inactivated through phosphorylation of specific residues including Ser99. Clinically, BAD phosphorylation has been reported to indicate poor survival and cisplatin resistance in ovarian cancer patients. Herein, we investigated the therapeutic potential of inhibiting BAD Ser99 phosphorylation in ovarian cancer.Material and methodsNPB, a novel small molecule which specifically inhibits BAD Ser99 phosphorylation, has been developed in our laboratory. Cell function assays performed include Alamar Blue cell viability assay, caspase3/7 assay, PI-Annexin V apoptosis assay, cell cycle analysis and 3D growth in Matrigel. The CI values are calculated by CompuSyn using Chou-Talalay method. The cancer stem cell-like cell population was examined by ALDEFLOUR and sphere formation assays.Results and discussionsThe level of BAD Ser99 phosphorylation is negatively correlated with cisplatin sensitivity in a panel of ovarian cancer cell lines. BAD Ser99 phosphorylation is increased upon acute cisplatin treatment of both sensitive and resistant ovarian cancer cells. The inhibition of BAD Ser99 phosphorylation by NPB alone increased apoptosis, and decreased cell viability, 3D growth, and anchorage-independent growth of both parental and cisplatin resistant ovarian cancer cells. In particular, NPB increased the sensitivity of the parental cells, and partially re-sensitised cisplatin resistant ovarian cancer cells towards cisplatin, with the combination of NPB and cisplatin showing a synergistic effect. An upstream BAD kinase, AKT has also been reported to mediate cisplatin resistance in ovarian cancer cells. Correspondingly, the combination of NPB and AKT inhibitor AZD5363 exhibited strong synergistic effects in both parental and cisplatin resistant cell lines. We also demonstrated that both the phosphorylation of BAD and its upstream kinase AKT was increased in this CSC-like population. NPB treatment alone was observed to decrease the CSC-like population, while the combination of AZD5363 and NPB produced a synergistic decrease in the CSC-like population.ConclusionOur preclinical data suggests that NPB, as a novel inhibitor of BAD Ser99 phosphorylation, can potentially be used in combination with cisplatin as a therapy for naïve ovarian cancer patients to increase cisplatin sensitivity. Furthermore, the combination of NPB and AKT inhibitor AZD5363 is a potential therapeutic approach in the treatment of cisplatin resistant ovarian cancer.

Efficacy of inhibition of BAD Ser99 phosphorylation by a novel small molecule in cisplatin resistant ovarian cancer

Event Abstract Back to Event Efficacy of inhibition of BAD Ser99 phosphorylation by a novel small molecule in cisplatin resistant ovarian cancer Yanxin Wang1* 1 National University of Singapore, Singapore Background Human BAD is a pro-apoptotic Bcl-2 family member, whose apoptotic functions can be inactivated through phosphorylation of specific residues including Ser99. Clinically, BAD phosphorylation has been reported to indicate poor survival and cisplatin resistance in ovarian cancer patients. Methods NPB, a novel small molecule which specifically inhibits BAD Ser99 phosphorylation, has been developed in our laboratory. Cell function assays performed include Alamar Blue cell viability assay, caspase3/7 assay, PI-Annexin V apoptosis assay, and 3D growth in Matrigel. The CI values are calculated using Chou-Talalay method. The cancer stem cell-like cell population was examined by ALDEFLOUR and sphere formation assays. Results The level of BAD Ser99 phosphorylation is negatively correlated with cisplatin sensitivity in a panel of ovarian cancer cell lines and it is increased upon acute cisplatin treatment in ovarian cancer cells. The inhibition of BAD Ser99 phosphorylation by NPB alone increased apoptosis, decreased anchorage-independent growth of both parental and cisplatin resistant ovarian cancer cells, with the combination of NPB and cisplatin showing a synergistic effect. An upstream BAD kinase, AKT has also been reported to mediate cisplatin resistance. Correspondingly, the combination of NPB and AKT inhibitor AZD5363 exhibited strong synergistic effects in both parental and cisplatin resistant cell lines. Both the phosphorylation of BAD and its upstream kinase AKT was increased in this CSC-like population. NPB treatment alone was observed to decrease the CSC-like population, while the combination of AZD5363 and NPB produced a synergistic decrease in the CSC-like population. Conclusion NPB, as a novel inhibitor of BAD Ser99 phosphorylation, can potentially be used in combination with cisplatin as a therapy for naïve ovarian cancer patients to increase cisplatin sensitivity. Furthermore, the combination of NPB and AKT inhibitor AZD5363 is a potential therapeutic approach in the treatment of cisplatin resistant ovarian cancer. Keywords: Bad phosphorylation, cisplatin resistance, ovarian cancer, NPB, Akt inhibitor Conference: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”, Putrajaya, Malaysia, 3 Dec - 5 Feb, 2019. Presentation Type: Speed Presentation Topic: Cancer Citation: Wang Y (2019). Efficacy of inhibition of BAD Ser99 phosphorylation by a novel small molecule in cisplatin resistant ovarian cancer. Front. Pharmacol. Conference Abstract: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”. doi: 10.3389/conf.fphar.2018.63.00013 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 07 Nov 2018; Published Online: 17 Jan 2019. * Correspondence: Ms. Yanxin Wang, National University of Singapore, Singapore, Singapore, wangyanxin98@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Yanxin Wang Google Yanxin Wang Google Scholar Yanxin Wang PubMed Yanxin Wang Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Extracellular vesicles from human liver stem cells restore argininosuccinate synthase deficiency

BackgroundArgininosuccinate synthase (ASS)1 is a urea cycle enzyme that catalyzes the conversion of citrulline and aspartate to argininosuccinate. Mutations in the ASS1 gene cause citrullinemia type I, a rare autosomal recessive disorder characterized by neonatal hyperammonemia, elevated citrulline levels, and early neonatal death. Treatment for this disease is currently restricted to liver transplantation; however, due to limited organ availability, substitute therapies are required. Recently, extracellular vesicles (EVs) have been reported to act as intercellular transporters carrying genetic information responsible for cell reprogramming. In previous studies, we isolated a population of stem cell-like cells known as human liver stem cells (HLSCs) from healthy liver tissue. Moreover, EVs derived from HLSCs were reported to exhibit regenerative effects on the liver parenchyma in models of acute liver injury. The aim of this study was to evaluate whether EVs derived from normal HLSCs restored ASS1 enzymatic activity and urea production in hepatocytes differentiated from HLSCs derived from a patient with type I citrullinemia.MethodsHLSCs were isolated from the liver of a patient with type I citrullinemia (ASS1-HLSCs) and characterized by fluorescence-activated cell sorting (FACS), immunofluorescence, and DNA sequencing analysis. Furthermore, their differentiation capabilities in vitro were also assessed. Hepatocytes differentiated from ASS1-HLSCs were evaluated by the production of urea and ASS enzymatic activity.EVs derived from normal HLSCs were purified by differential ultracentrifugation followed by floating density gradient. The EV content was analyzed to identify the presence of ASS1 protein, mRNA, and ASS1 gene. In order to obtain ASS1-depleted EVs, a knockdown of the ASS1 gene in HLSCs was performed followed by EV isolation from these cells.ResultsTreating ASS1-HLSCs with EVs from HLSCs restored both ASS1 activity and urea production mainly through the transfer of ASS1 enzyme and mRNA. In fact, EVs from ASS1-knockdown HLSCs contained low amounts of ASS1 mRNA and protein, and were unable to restore urea production in hepatocytes differentiated from ASS1-HLSCs.ConclusionsCollectively, these results suggest that EVs derived from normal HLSCs may compensate the loss of ASS1 enzyme activity in hepatocytes differentiated from ASS1-HLSCs.

Abstract 3923: Oncogenic kinase-induced PKM2 Y105 phosphorylation promotes tumorigenesis and cancer stem cell-like property

Abstract The role of pyruvate kinase M2 isoform (PKM2) in tumor progression is controversial. Previously, it was reported that PKM2 has an oncogenic function in promoting tumor growth in xenograft models; however, PKM2 was indicated non-oncogenic or even anti-oncogenic because depletion of PKM2 accelerates mammary tumor growth in the Brca1 knockout mouse model of breast cancer. Since tumors frequently harbor various abnormally activated oncogenic kinases, we hypothesized that phosphorylation of PKM2 by oncogenic kinases in tumor cells enables PKM2 to promote tumor growth, while unphosphorylated PKM2 in non-transformed cells cannot promote tumor growth. Here, we report that PKM2 proteins were largely unphosphorylated and formed tetramers in normal mammary epithelial cells (MCF10A, MCF12A). On the other hand, PKM2 proteins were phosphorylated at Y105 and formed dimers in breast cancer cells (MDA-MB-231, MDA-MB-435). Knocking down PKM2 didn’t affect normal mammary epithelial cell (MCF10A) growth but significantly decreased breast cancer cell MDA-MB-231 proliferation. In addition, introducing the PKM2-Y105D phosphomimetic mutant into MCF10A cells induced epithelial to mesenchymal transition, increased CD44Hi/CD24Neg stem cell-like cell population, and induced colony formation in soft agar, promoted cell migration and invasion. Interestingly, PKM2-Y105D increased YAP1 protein nuclear localization in MCF10A cells, whereas inhibiting YAP1 by siRNA or a chemical inhibitor (Verteporfin) decreased PKM2-Y105D-induced cancer stem cell-like population and colony formation ability. The data indicate that YAP1 is a novel downstream target of pY105-PKM2 in promoting the stem-like property and triggering mammary tumor initiation. Next, we performed receptor tyrosine kinase array and identified several oncogenic tyrosine kinases (AXL, EPHA2, FAK, Src, JAK3 and so on) that effectively phosphorylates PKM2 at Y105. Currently, we are testing the efficacy of targeting these oncogenic tyrosine kinases in reversing the phosphorylation of PKM2 and blocking its tumor-promoting function. Together, our data indicate that oncogenic kinases-induced phospho-Y105-PKM2, but not the unphosphorylated PKM2, promotes mammary tumor initiation by increasing cancer stem cell populations. Citation Format: Zhifen Zhou, Min Li, Lin Zhang, Dihua Yu. Oncogenic kinase-induced PKM2 Y105 phosphorylation promotes tumorigenesis and cancer stem cell-like property [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3923. doi:10.1158/1538-7445.AM2017-3923

Embryonic Stem Cell-Like Population in Dupuytren's Disease Expresses Components of the Renin-Angiotensin System.

3,3-Diaminobenzidine (DAB) and immunofluorescent immunohistochemical (IHC) staining for (pro)renin receptor (PRR), angiotensin-converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1), and angiotensin II receptor 2 (ATIIR2) was performed on 4-μm thick formalin-fixed paraffin-embedded sections of DD cords and nodules from 6 patients. Western blotting (WB) and NanoString mRNA analysis were performed to confirm RAS protein expression and transcriptional activation, respectively. IHC staining demonstrated the expression of PRR, ACE, ATIIR1, and ATIIR2 on the ERG+ and CD34+ endothelium of the micro vessels surrounding the DD cords and nodules. PRR was also expressed on the pericyte layer of these microvessels. WB confirmed protein expression of PRR, ACE, and ATIIR2 but not ATIIR1. NanoString analysis confirmed transcriptional activation of PRR, ACE, ATIIR1, but ATIIR2 was below detectable levels. We demonstrated expression of PRR, ATIIR1, ATIIR2, and ACE on the embryonic stem cell-like cell population on the microvessels surrounding DD nodules and cords by IHC staining, although the expression of ATIIR1 was not confirmed by WB and that of ATIIR2 was below detectable levels on NanoString analysis. These findings suggest the embryonic stem cell-like cell population as a potential therapeutic target for DD, by using RAS modulators.

Identification of EmSOX2, a member of the Sox family of transcription factors, as a potential regulator of Echinococcus multilocularis germinative cells

Larvae of the tapeworm Echinococcus multilocularis cause alveolar echinococcosis (AE), one of the most lethal helminthic infections in humans. The germinative cells, a population of stem cell-like cells, are considered to drive the continuous growth of the metacestodes within the host. The mechanisms and relative molecules controlling the behavior of germinative cells are poorly understood. Sox transcription factors play important roles in maintenance and regulation of stem/progenitor cells. We here describe the identification of a Sox family member in E. multilocularis, EmSOX2, as a potential regulator of germinative cells. Replacement of mouse Sox2 with EmSox2 could derive induced pluripotent stem cells (iPSCs) from somatic cells, suggesting that EmSOX2 is functionally related to mammalian SOX2. EmSOX2 is actively expressed in the proliferating germinative cells in E. multilocularis, and is significantly downregulated upon specific depletion of the germinative cell population by hydroxyurea treatment. These findings suggest that EmSOX2 may play a critical role in regulating E. multilocularis germinative cells.

EGF-mediated EGFR/ERK signaling pathway promotes germinative cell proliferation in Echinococcus multilocularis that contributes to larval growth and development

BackgroundLarvae of the tapeworm E. multilocularis cause alveolar echinococcosis (AE), one of the most lethal helminthic infections in humans. A population of stem cell-like cells, the germinative cells, is considered to drive the larval growth and development within the host. The molecular mechanisms controlling the behavior of germinative cells are largely unknown.Methodology/Principal findingsUsing in vitro cultivation systems we show here that the EGFR/ERK signaling in the parasite can promote germinative cell proliferation in response to addition of human EGF, resulting in stimulated growth and development of the metacestode larvae. Inhibition of the signaling by either the EGFR inhibitors CI-1033 and BIBW2992 or the MEK/ERK inhibitor U0126 impairs germinative cell proliferation and larval growth.Conclusions/SignificanceThese data demonstrate the contribution of EGF-mediated EGFR/ERK signaling to the regulation of germinative cells in E. multilocularis, and suggest the EGFR/ERK signaling as a potential therapeutic target for AE and perhaps other human cestodiasis.

Triple-negative breast cancers with amplification of JAK2 at the 9p24 locus demonstrate JAK2-specific dependence.

Amplifications at 9p24 have been identified in breast cancer and other malignancies, but the genes within this locus causally associated with oncogenicity or tumor progression remain unclear. Targeted next-generation sequencing of postchemotherapy triple-negative breast cancers (TNBCs) identified a group of 9p24-amplified tumors, which contained focal amplification of the Janus kinase 2 (JAK2) gene. These patients had markedly inferior recurrence-free and overall survival compared to patients with TNBC without JAK2 amplification. Detection of JAK2/9p24 amplifications was more common in chemotherapy-treated TNBCs than in untreated TNBCs or basal-like cancers, or in other breast cancer subtypes. Similar rates of JAK2 amplification were confirmed in patient-derived TNBC xenografts. In patients for whom longitudinal specimens were available, JAK2 amplification was selected for during neoadjuvant chemotherapy and eventual metastatic spread, suggesting a role in tumorigenicity and chemoresistance, phenotypes often attributed to a cancer stem cell-like cell population. In TNBC cell lines with JAK2 copy gains or amplification, specific inhibition of JAK2 signaling reduced mammosphere formation and cooperated with chemotherapy in reducing tumor growth in vivo. In these cells, inhibition of JAK1-signal transducer and activator of transcription 3 (STAT3) signaling had little effect or, in some cases, counteracted JAK2-specific inhibition. Collectively, these results suggest that JAK2-specific inhibitors are more efficacious than dual JAK1/2 inhibitors against JAK2-amplified TNBCs. Furthermore, JAK2 amplification is a potential biomarker for JAK2 dependence, which, in turn, can be used to select patients for clinical trials with JAK2 inhibitors.

Abstract 4241: ONC201/TIC10 targets colorectal cancer stem cells and bulk tumor cells via an Akt-Foxo3a-TRAIL-dependent mechanism

Abstract ONC201/TIC10 is a first-in-class anti-tumor agent that induces cell death selectively in cancer cells in vitro and in vivo. ONC201/TIC10 is entering phase I/II clinical trials for patients with advanced cancer. Colorectal tumors contain a small population of stem cell-like cells (cancer stem cells, CSCs) capable of self-renewal that contributes to tumor maintenance, resistance to therapy and disease relapse. We hypothesized that the potent anti-tumor effect of ONC201/TIC10 in colon cancer involves targeting both CSCs and bulk tumor cells. We show that ONC201/TIC10 depletes CSC markers, prevents CSC-mediated colonosphere formation and induces cell surface TRAIL in CSCs in vitro. We further validated these results in xenograft tumor studies in mice, compared the effects of ONC201/TIC10 in CSCs and non-CSCs and validated the mechanism of TRAIL induction in CSCs. ONC201/TIC10 significantly reduces the growth of tumors initiated with sorted Aldefluor(+) CSCs and prevents the passage of these tumors. ONC201/TIC10 upregulates apoptosis markers cleaved caspase-8 and cleaved PARP and downregulates CSC markers ALDH, CD44 and CD133 in the xenograft tumors. In a limiting dilution xenograft tumor initiation assay ONC201/TIC10-treatment decreased tumor initiation and was superior to 5-Fluorouracil treatment. Thus, ONC201/TIC10 inhibits CSC self-renewal in vivo. We have previously described that ONC201/TIC10 exposure leads to transcriptional induction of the TRAIL gene via the transcription factor Foxo3a, which is activated by dual inactivation of Akt and ERK. ONC201/TIC10 reduces levels of phosho-Akt, phospho-ERK, phospho-S294 Foxo3a, phospho-S253 Foxo3a and induces TRAIL in CSC-initiated xenograft tumors. ONC201/TIC10-mediated effects on Akt, ERK, Foxo3a and TRAIL occur in both bulk tumor cells and CSCs. Next, we performed control experiments to confirm the Akt-Foxo3a-TRAIL-dependent mechanism. ONC201/TIC10-mediated depletion of CD44(+) cells was significantly blocked by the TRAIL-sequestering antibody RIK-2. We have previously reported that ONC201/TIC10 upregulates DR5 in tumor cells and disruption of the DR5 death domain decreases ONC201/TIC10-mediated cell death. ONC201/TIC10 increases cell surface DR5 in CSCs and non-CSCs. DR5 knockdown rescues ONC201/TIC10-mediated colonosphere inhibition and depletion of CD44(+) cells further supporting a DR5-TRAIL-dependent mechanism. Overexpression of myristoylated Akt significantly reduces induction of surface TRAIL and rescues depletion of CSC markers and colonosphere inhibition by ONC201/TIC10. Foxo3a knockdown also significantly rescues ONC201/TIC10-mediated colonosphere inhibition and depletion of CD44(+) cells. The identification and demonstration of CSCs as a target further supports the use of ONC201/TIC10 for colorectal cancer therapy and provides an innovative biomarker that could be monitored in the clinic. Citation Format: Varun Vijay Prabhu, Joshua E. Allen, David T. Dicker, Wafik S. El-Deiry. ONC201/TIC10 targets colorectal cancer stem cells and bulk tumor cells via an Akt-Foxo3a-TRAIL-dependent mechanism. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4241. doi:10.1158/1538-7445.AM2015-4241

Abstract 1254: Mitochondrial SOD2 regulates EMT and cancer stem cell-like cell populations

Abstract Epithelial-mesenchymal transition (EMT) is involved in cancer cell invasion, metastasis and treatment failure. EMT may promote conversion of a subset of cancer cells defined by low expression of CD44 (CD44L) to those with high expression of CD44 (CD44H), the latter associated with increased malignant properties of cancer cells. Triggered by a variety of stimuli such as transforming growth factor (TGF)-β, hypoxia and chemoradiation, EMT may be activated in cancer cells via stress-induced reactive oxygen species (ROS) in the tumor microenvironment. Excessive ROS may cause cell death or senescence; however, cells undergoing EMT may require a physiological level of ROS. It remains elusive as to how cellular antioxidant capabilities may influence EMT. Mitochondrial superoxide dismutase 2 (SOD2) is frequently overexpressed in oral and esophageal cancers. Here, we investigated mechanisms of SOD2 transcriptional regulation in EMT as well as the functional role of this antioxidant in EMT. Using well-characterized genetically engineered oral and esophageal human epithelial cell lines coupled with RNA interference (RNAi) and flow cytometric approaches, we find that TGF-β stimulates EMT, resulting in conversion of CD44L to CD44H cells, the latter of which display SOD2 upregulation. SOD2 induction in transformed cells was concurrent with suppression of TGF-β-mediated induction of both ROS and senescence. SOD2 gene expression appeared to be transcriptionally regulated by NF-κB and ZEB2, but not ZEB1. Moreover, SOD2-mediated antioxidant activity may restrict conversion of CD44L cells to CD44H cells at the early stages of EMT. In aggregate, this study provides novel mechanistic insights into the dynamic expression of SOD2 during EMT and delineates a functional role for SOD2 in EMT, influencing distinct CD44L and CD44H subsets of cancer cells that have been implicated in oral and esophageal tumor biology. Note: This abstract was not presented at the meeting. Citation Format: Kelly A. Whelan, Hideaki Kinugasa, Koji Tanaka, Satish Srinivasan, Manti Guha, Daret St. Clair, Andres Klein-Szanto, Narayan Avadhani, Alan Diehl, Anil Rustgi, Hiroshi Nakagawa. Mitochondrial SOD2 regulates EMT and cancer stem cell-like cell populations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1254. doi:10.1158/1538-7445.AM2015-1254

Notch3 interactome analysis identified WWP2 as a negative regulator of Notch3 signaling in ovarian cancer.

The Notch3 signaling pathway is thought to play a critical role in cancer development, as evidenced by the Notch3 amplification and rearrangement observed in human cancers. However, the molecular mechanism by which Notch3 signaling contributes to tumorigenesis is largely unknown. In an effort to identify the molecular modulators of the Notch3 signaling pathway, we screened for Notch3-intracellular domain (N3-ICD) interacting proteins using a human proteome microarray. Pathway analysis of the Notch3 interactome demonstrated that ubiquitin C was the molecular hub of the top functional network, suggesting the involvement of ubiquitination in modulating Notch3 signaling. Thereby, we focused on functional characterization of an E3 ubiquitin-protein ligase, WWP2, a top candidate in the Notch3 interactome list. Co-immunoprecipitation experiments showed that WWP2 interacted with N3-ICD but not with intracellular domains from other Notch receptors. Wild-type WWP2 but not ligase-deficient mutant WWP2 increases mono-ubiquitination of the membrane-tethered Notch3 fragment, therefore attenuating Notch3 pathway activity in cancer cells and leading to cell cycle arrest. The mono-ubiquitination by WWP2 may target an endosomal/lysosomal degradation fate for Notch3 as suggested by the fact that the process could be suppressed by the endosomal/lysosomal inhibitor. Analysis of The Cancer Genome Atlas dataset showed that the majority of ovarian carcinomas harbored homozygous or heterozygous deletions in WWP2 locus, and there was an inverse correlation in the expression levels between WWP2 and Notch3 in ovarian carcinomas. Furthermore, ectopic expression of WWP2 decreased tumor development in a mouse xenograft model and suppressed the Notch3-induced phenotypes including increase in cancer stem cell-like cell population and platinum resistance. Taken together, our results provide evidence that WWP2 serves as a tumor suppressor by negatively regulating Notch3 signaling in ovarian cancer.

Abstract 3868: Six1 overexpression promotes malignant progression in models of cervical and colon cancer

Abstract The overexpression of Six1, a member of the Six family of homeodomain transcription factors, has been found in various human cancers, and is associated with increased tumor progression and metastasis. Previous studies in our laboratory discovered that the expression of Six1 mRNA increased during in vitro progression of human papillomavirus type 16 (HPV16) immortalized human keratinocytes (HKc/HPV16) toward a differentiation-resistant (HKc/DR) phenotype. The overexpression of Six1 induced Epithelial-Mesenchymal Transition (EMT) by the activation of p38 MAPK in HKc/DR. Although immortalized and resistant to serum and calcium-induced differentiation, HKc/DR are not tumorigenic. To further explore the role of Six1 in HPV16 immortalized cells we determined whether overexpression of Six1 in HKc/DR resulted in malignant conversion. We found that HKc/DR overexpressing Six1 (DR-Six1), but not HKc/DR controls (DR-Ctrl), formed squamous cell carcinomas in nude mice. Cancer stem cells are proposed to drive tumor initiation and progression; for this reason we analyzed cancer stem cell markers in DR-Ctrl and DR-Six1. We found that DR-Six1 had an increased CD24-/CD44+ population and increased aldehyde dehydrogenase 1 (ALDH1) expression as compared to DR-Ctrl, indicating a cancer stem cell-like phenotype in Six1 overexpressing cells. We then used the human cervical cancer cell line HeLa to further explore the enhanced tumorigenic and malignant properties associated with Six1 overexpression. We determined that overexpressing Six1 in HeLa increased their ability to form spheroids in vitro and increased tumor size in vivo. As an extension of this work, we investigated the role of Six1 on tumorigenesis and metastasis in colon cancer in vivo and in vitro by using the murine colon cancer cell line MC38. We found that Six1 promoted colon cancer tumorigenesis, progression and metastasis, and increased the population of cancer stem cell-like cells. Taken together, our studies demonstrate that Six1 overexpression increases the cancer stem cell-like cell population, and promotes malignant progression in models of cervical and colon cancer. Supported by grant P20MD001770 from the National Institute on Minority Health and Health Disparities. Citation Format: Hanwen Xu, Yu Zhang, Lucia Pirisi, Maria Marjorette Pena, Kim E. Creek. Six1 overexpression promotes malignant progression in models of cervical and colon cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3868. doi:10.1158/1538-7445.AM2014-3868

Characterisation of adrenocortical progenitor cells: isolation of mesenchymal stem cell-like cell populations from human adrenal cortex

Characterisation of adrenocortical progenitor cells: isolation of mesenchymal stem cell-like cell populations from human adrenal cortex

How prostate-specific membrane antigen level may be correlated with stemness in prostate cancer stem cell-like cell populations?

Prostate-specific membrane antigen (PSMA) is a widely used targeted molecule in prostate patients. The present research, attempts to support the hypothesis that PSMA expression in prostate cancer stem cell-like (CSC) cell populations may be correlated with nanog and other transcription factors in different stages of prostate carcinomas. To provide more accurate evidence of the above, a population of prostate CSCs was isolated and analyzed using different protocols. The first method was based in the ability of CSCs to form spherical colonies in semi-suspension of a culture. A qPCRbased protocol and a flow cytometric analysis protocol were chosen to test the presence of stemness markers and PSMA in the selected populations. The formation of micro-sphere in semi-suspension has been pointed out. In the other panels of the test, the linear correlation between PSMA and nanog in gene and protein level was shown. However, the statistical analysis including the coefficient of variationand standard deviation's values) has proved that there were differences in PSMA expression between cancer cells and CSCs. The previous analysis has pointed out that PSMA expression may be correlated with nanog's expression as well as with other confounders in a population of prostate CSCs.

Abstract 4771: Regulatory role of an orphan nuclear receptor LRH-1 in castration-resistant growth of prostate cancer cells.

Abstract The advanced development of castration-resistant prostate cancer (CRPC) in patients upon androgen-deprivation therapy is generally believed to be mostly mediated by reactivation of androgen receptor (AR) signaling or its bypass with mechanisms involved, including clonal selection of androgen-independent or stem cell-like cell populations, AR hypersensitivity due to AR overexpression, and ligand promiscuity or independence by AR mutations, intratumoral conversion of adrenal androgens to high affinity AR ligands. Besides, the de novo steroid synthesis from cholesterol in CRPC has also been proposed. In this study, we found that the nuclear receptor liver receptor homolog-1 (LRH-1) might contribute to the CRPC growth or de novo steroid synthesis in prostate cancer cells through its positive regulation on expression of several critical enzymes in steroidogenesis, including CYP17A1, HSD3B1, HSD3B2 and StAR. Functional analyses showed that overexpression of LRH-1 could induce higher resistance to antiandrogen bicalutamide and steroid-depleted culture condition in AR-positive androgen-sensitive (LNCaP) but not in AR-negative androgen-insensitive (DU145) prostate cancer cells. In vivo tumorigenicity study showed that LNCaP-LRH-1 cells grew more aggressively in castrated or intact SCID mice, contrary to LNCaP-vector control cells that did not grow in castrated mice. Importantly, liquid chromatography-tandem mass spectrometry showed that intratumoral androgen concentrations (testosterone and DHT) were significantly higher in tumors formed by LNCaP-LRH-1 cells than those by LNCaP-vector control cells. Treatment of LNCaP-LRH-1 cells with a CYP17A1 inhibitor abiraterone could restore their sensitivity to bicalutamide and steroid-depleted culture condition. Gene knockdown of endogenous LRH-1 in AR-positive VCaP prostate cancer cells could not only suppress the mRNA expression of steroidogenic enzymes but also enhance their sensitivity to antiandrogen. Together, our results indicated that LRH-1 might play a regulatory role in advanced castration-resistant growth of prostate cancer via positive control on de novo steroid synthesis in prostate cancer cells. (This work was supported by a Direct Grant for Research 2011/2012 and a RGC-GRF grant 2009/2010) Citation Format: Lijia Xiao, Shan Yu, Wendy W.L. Hsiao, Franky Leung Chan. Regulatory role of an orphan nuclear receptor LRH-1 in castration-resistant growth of prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4771. doi:10.1158/1538-7445.AM2013-4771